Telomeres are the DNA structure at the ends of the chromosomes of eukaryotes, including human, and are comprised of variable lengths of double strander repeats terminating with single-stranded repeats originally identified in yeast and protozoa (Makarov et al., 1997, Cell 88:657-666).
Review articles concerning telomeres include Greider, 1996, Ann. Rev. Biochem. 65:337 and Zakian, 1995, Science 270:1601. Relevant articles on various aspects of telomeres include Cooke and Smith, 1986, Cold Spring Harbor Symp. Quant. Biol. 51:213; Morin, 1989, Cell 59:521; Blackburn et al., 1989, Genome 31:553; Szostak, 1989, Nature (London) 337:303; Gall, 1990, Nature (London) 344:108; Henderson et al., 1990, Biochemistry 9:732; Gottschling et al., 1990, Cell 630:751; Harrington et al., 1991, Nature (London) 353:451; Muller et al., 1991, Cell .sctn.67:815; Yu et al., 1991, Cell 67:823; Gray et al., 1991, Cell 67:807; de Lange, 1995, "Telomere Dynamics and Genome Instability in Human Cancer", E. Blackburn and C. W. Greider (eds), in Telomeres, Cold Spring Harbor Laboratory Press, pp. 265-293; Rhyu, 1995, J. Natl. Cancer Inst 87:884; Greider and Harley, 1996, "Telomeres and Telomerase in Cell Senescence and Immortalization", in Cellular Aging and Cell Death, Wiley-Liss, Inc., pp. 123-138. Other articles of some relevance include Lundblad et al., 1989, Cell 57:633 and Yu et al., 1990, Nature (London) 344:126.
Maintenar the integrity of teloees is essential for cell suival (Muller, 1938, The Collectirg Net 13:181-195; Sandell et al., 1993, Cell 75:729-739). The proliferative potential of cells has been correlated with alterations in the length of these tandemly repeated sequences (Zakian, 1989, Ann. Rev. Genet 23:579-604; Counter et al., 1992, EMBO J. 11:1921-1929).
The finite replicative capacity of normal human cells, e.g., fibroblasts, is characterized by a cessation of proliferation in spite of the presence of serum growth factors. This cessation of replication after a maximum of 50 to 100 population doublings in vitro is referred to as cellular senescence. See, Goldstein, 1990, Science 249:1129; Hayflick and Moorehead, 1961, Exp. Cell Res. 25.585; Hayflick, 1985, ibid., 37:614; Ohno, 1979, Mech. Aging. Dev. 11:179; Ham and McKeehan, 1979, "Media and Growth Requirements", W. B. Jacoby and I. M. Pastan (eds), in Methods of Enzymology, Academic Press, NY, 58:44-93. The replicative life span of cells is inversely proportional to the in vivo age ofthe donor (Martin et al., 1979, Lab. Invest. 23:86; Goldstein et al. 1969, Proc. Natl. Acad. Sci. USA 64:155; Schneider and Mitsui, 1976, ibid, 73:3584) and is therefore suggested to reflect in vivo ageing on a cellular level.
Cellular immortalization (unlimited life span) may be thought of as an abnormal escape from cellular senescence (Shay et al., 1991, Emp. Cell Res. 196:33). Normal human somatic cells appear to be mortal, i.e., have finite replication potential. In contrast, the germ line and malignant tumor cells are immortal (have indefinite proliferative potential). Human cells cultures in vitro appear to require the aid of transforming oncoproteins to become immortal and even then the frequency of immortalization is 10.sup.-6 to 10.sup.-7 (Shay et al., 1989, Emp. Cell Res. 184:109). A variety of hypotheses have been advanced over the years to explain the causes of cellular senescence. One such hypothesis proposes that the loss of telomec DNA with age, eventually triggers cell cycle exit and cellular senescence (Zakian, 1989, Ann. Rev. Genet 23:579-604; Harley et al. 1990, Nature (London) 345:458-460; Hastie et al., 1990, Nature (London) 346:866-868; Allsopp et al., 1992, Proc, Natl. Acad. Sci. USA 89:10114-10118; Counter et al., 1992, EMBO J. 11:1921-1929).
Human primary fibroblasts in culture enter crisis after a precise number of cell division associated with gradual telomere shortening, at which point all the cells die (de Lange, 1994, Proc. Natl. Acad. Sci. USA 91:2882-2885). Mouse primary fibroblasts have longer and/or more stable telomeres and display a similar behavior when cultured in vitro (Prowse and Greider, 1994, Proc. Natl. Acad. Sci. USA, 92:4818-4822). However, after crisis, primary mouse cells in culture spontaneously immortalize with a frequency of 10.sup.-6, possibly because longer telomeres facilitate the growth of mutant cells (de Lange, 1994, Proc. Natl. Acad. Sci. USA 91:2882-2885).
It should be noted, as mentioned above, that other hypotheses have been advanced to explain senescence and that there is yet to be a consensus or a universally accepted hypothesis therefor. Previously, the causal relationship between telomeres and cancer/ageing/senescence had been built entirely on correlative studies.
Recent data has shown that telomeres play a direct role in cell senescence and transformation. Indeed, Wright et al., 1996, EMBO J. 15:1734-1741, using telomerase-negative cells which have limited life span in tissue culture, have shown that the introduction of oligonucleotides carrying telomeric repeats causes telomere elation and increases the proliferative capacity of these cells. Moreover, the authors state that "previous studies had shown a remarkable correlation between telomere length and cellular senescence. The present results provide the first experimental evidence for a true causal relationship between telomere length and a limited proliferative capacity". Feng et al., 1995, Science 269:1236-1241 showed that human cell line (HeLa) transfected with an antsense telomere RNA, loose telomeric DNA and begin to die after 23-26 cell doublings. The author claim that "the results support the hypothesis that telomere loss leads to crisis and cell death once telomeres are shortened to a critical length".
The postulated link between senescence/proliferation of cells and telomere length has led to therapeutic and diagnostic methods relating to telomere length or to telomerase, the ribonucleoprotein enzyme involved in the synthesis of telomeric DNA. PCT Publication No. 93/23572 describes oligonucleotide agents that either reduce the loss of telomeric sequence during passage of cells in vitro, or increase telomeric length of immortal cells in vitro. The same type of approach is also taught in PCT Publication No. 94/13383 and U.S. Pat. No. 5,484,508 which refer to methods and compositions for the determination of telomere length and telomerase activity, as well as to methods to inhibit telomerase activity in the treatment of proliferative diseases. Methods to increase or decrease the length of telomeres through an action on telomerase is also taught. The agents which are shown to reduce telomere loss of telomere length during proliferation are oligonucleotides which promote synthesis of DNA at the telomere ends, as well as telomerase.
PCT Publication No. 95/13383 discloses a method and compositions for increasing telomeric length in normal cells so as to increase the proliferative capacity of the cells and to delay the onset of cellular senescence. PCT Publication No. 96/10035 teaches that telomere length serves as a biomarker for cell turnover. Furthermore, it discloses that measuemert of telomere length can be used to diagnose and stage cancer and other diseases as well as cell senescence.
Heterogeneous nuclear ribonucleoprotein particles (hnRNP) proteins are abundant proteins mammalian cells, of which the A to U members have been best characterized due to their RNA binding properties (Dreyfuss et al., 1993, Ann. Rev. Biochem. 62:289). There are over 20 such hnRNP proteins in human cells. The best characterized hnRNP protein so far is the hnRNP A1 protein which has been shown to be involved in alternative RNA splicing (Mayeda et al., 1992, Cell 68:365-375; Yang et al., Proc. Natl. Acad. Sci. USA. 91:6924-6928). Indeed, the hnRNP A1 protein has high affinity for RNA in vitro (Bird and Dreyfuss, 1994, EMBO J. 13:1197-1204). UP1 is a proteolytic product derived from hnRNP A1 (Riva et al., 1986, EMBO J. 5:2267-2273). UPI has no activity in alternative splicing in vitro (Mayeda et al., 1994, EMBO J. 13:5483-5495). In vitro experiments have shown that hnRNP A1 binds to oligonucleotides containing vertebrate 3' splice site sequences (Buvoli et al., 1990, Nucl. Acids Res. 18:6595). The DNA version of 3' splice site sequences share some similarity with vertebrate telomeric repeats (Ishikawa et al., 1993, Molec. Cell. Biol. 13:4301-4310). In vitro data concerning hnRNP A1 (UP1) can be summarized as follows: (1) A1 binds to DNA oligonucleotides carrying 3' splice site sequences which contain TTAGGT(Buvoli et al., 1990, Nucl. Acids Res. 18:6595); (2) UP1 is part of complexes assembled on oligonucleotides carrying telomeric repeats (TTAGGG)n (Ishikawa et al., 1993, Molec. Cell. Biol. 13:4301-4310), indicating that A1 and/or UP1 "could perhaps" bind to chromosome telomeres. However, this in vitro result is not yet correlated with in vivo data and cannot demonstrate th direct interaction of A1 and/or UP1 with telomeric repeats in the complex formed. Moreover, the oligonucleotides used in the expement which carry telomeric repeats also resemble the 3' splice site oligo used by Buvoli et al., 1990, Nucl. Acids Res. 18:6595. This could mean that (UP1) binds to 3' splice site sequences and that the telomeric sequence happens to resemble a 3' splice site (DNA version), and (3) Bird and Dreyfuss, 1994, EMBO J. 13:1197-1204 show that A1 binds to RNA and that the optimal sequence recognized by A1 resembles a 3' splice site and a 5' splice site. Although the optimal recognition sequence also resembles a telomeric repeat, the hypothesis that A1 might bind to telomeres appears to have been discarded. Models in which A1 binds to its preferred sequence in the context of an RNA molecule to modulate splicing, transport and possibly translation appear to be favoured (Bird and Dreyfuss, 1994, EMBO J. 13:1197-1204).
Proteins that bind telomeric repeats may be subdivided into two categories, those that bind double-stranded repeats and those that bind single-stranded repeats. See Lin, 1993, BioEssays 15:555. A number of proteins binding to double-stranded repeats have been characterized. These include RAP1 and TBF.alpha. from yeast, PPT in Physarum and TRF in mammals.
While TBF.alpha. and PPT do bind oligonucleotides containing telomeric sequences in vitro, there has been no demonstration that these proteins bind telomeres in vivo. RAP1, but not TBF.alpha., influences the length of telomeres in yeast (Conrad et al., 1990, Cell 63:739). In mammals, it is noteworthy that TRF has been shown to bind to telomeres in vitro (Chong et al., 1995, Science 270:1663). Moreover, overexpression of TRF promotes a reduction in the length of telomeres while expessing a mutated form of TRF causes telomere elongation (van Steensel et al., 1997, Nature (London) 385:740 and de Lange W097/08314).
Proteins that can bind to single-stranded telomeric repeats in vitro include protein .alpha. and .beta. of Oxytrichia and Stylonychia, a of Euplotes, MF3 of chicken and XTEF of Xenopus. There has been no demonstration that the vertebrate proteins MF3 and XTEF bind to telomeres in vivo and no suggestion that their expression influences the size of telomeres. The yeast NSR1, GBP2, cdc13/EST4 and EST1 proteins were shown to bind to single-stranded yeast telomeric DNA (Lin and Zakian, 1994, Nucl. Acids Res. 22:4906; Nugent et al., 1996, Science 274:249; Virta-Pearlman et al., 1996, Genes Dev. 10:3094). While NSR1 and GBP2 do not affect telomere length in vivo, mutant strains engineered not to exress cdc13p or to express mutated forms of EST1 undergo telomere attrition despite having wild-type amounts of telomerase activity (Nugent et al., 1996, Science 274:249; Virta-Pearlman et al., 1996, Genes Dev. 10:3094). A limited number of mammalian proteins, including hnRNP proteins, have been reported to associate with oligonucleotides carrying telomeric repeats (Ishikawa et al., 1993, Molec. Cell. Biol. 13:4301-4310; McKay and Cooke, 1992, Nucl. Acids Res. 20:6461-6464; de Lange, 1996, Seminars in Cell & Dev. Biol. 7:23-29; Sang et al., 1997, J. Biol. Chem. 272:4474-4482). Whether any of these proteins bind to single-stranded telomeric repeats in cells and affect telomere length in vivo has yet to be documented. In any event the observation that a protein binds to telomeric sequences in vitro cannot be considered predictive of a role on the size of telomeres in vivo.
There thus remains a need for reagents other than oligonucleotides and telomerase that can influence the length of telomeres in cells and for methods to increase or decrease the proliferative capacity of cells and to delay or precipitate the onset of senescence. The present invention seeks to meet these and other needs as described below.